Abstract The RAS/MAP kinase pathway is aberrantly activated in a wide variety of human cancers. The V600E mutation in BRAF, a kinase in this pathway, causes approximately one-half of all melanomas and is the driver in many other cancers as well. Despite decades of intense interest and investigation, BRAF regulation is not well- understood. Furthermore, compounds targeting the RAS/MAPK pathway exhibit poorly understood pharmacologic effects. BRAF inhibitors, such as vemurafenib, potently inhibit V600E BRAF, but they paradoxically activate wild type BRAF. Inhibitors of MEK, a kinase downstream of BRAF, differ in their efficacy depending upon whether the pathway is activated by mutations in KRAS versus BRAF. Collectively, the confusing pharmacology of these agents reflects our incomplete knowledge of the regulation and biochemical workings of this pathway and limits our ability to develop targeted therapies for BRAF and the RAS/MAPK pathway. Over the last two decades, my laboratory has focused on the structural biology of protein kinases and their dysregulation in cancer, and on cancer drug discovery. We have applied our basic biophysical, biochemical and structural insights into wild-type and mutant EGFR to discover new classes of pharmacologic agents targeting the mutant receptor, including both mutant-selective covalent and allosteric inhibitors that can overcome resistance mechanisms. We are now applying an analogous structural and mechanistic approach to demystify BRAF regulation and pharmacology. Our objectives are to understand BRAF regulation in structural detail, to decipher the complex pharmacology of the BRAF and MEK inhibitors, and to develop new agents that target the pathway in a mutant-selective manner. To achieve these goals, we will determine the structure of autoinhibited and active BRAF complexes using cryo-electron microscopy. We will reconstitute the pathway from KRAS to ERK using purified components in order to dissect mechanisms of BRAF and MEK activation and probe the effects of pharmacologic agents that target the pathway. In addition, we will use these reconstitutions together with our structural insights to discover new agents that target the pathway in a mutant-selective manner. These studies will provide fundamental new understanding of BRAF regulation and, in the long term, they should yield more effective and better tolerated therapies for cancers driven by mutagenic activation of this pathway.